1. Field of the Invention
The present invention relates to compounds, compositions, and methods of use for the inhibition of gene expression and/or activity or for diagnosing, treating and/or preventing diseases and/or conditions that respond to the inhibition of gene expression and/or activity.
2. Summary of the Related Art
An approach to inhibit gene expression is antisense technology or RNA inhibition (RNAi). These approaches make use of sequence-specific binding of DNA and/or RNA based oligonucleotides to selected mRNA, microRNA, preRNA or mitochondrial RNA targets and the inhibition of translation that results therefrom. This oligonucleotide-based inhibition of translation and ultimately gene expression is the result of one or more cellular mechanisms, which may include but is not limited to (i) direct (steric) blockage of translation, (ii) RNase H-mediated inhibition, and (iii) RNAi-mediated inhibition (e.g. short interfering-RNA (siRNA), microRNA (miRNA), DNA-directed-RNAi (ddRNAi), and single-stranded RNAi (ssRNAi)).
The history of antisense technology has revealed that while determination of antisense oligonucleotides that bind to mRNA is relatively straight forward, the optimization of antisense oligonucleotides that have true potential to inhibit gene expression and therefore be good clinical candidates is not. Accordingly, if an oligonucleotide-based approach to down-regulating gene expression is to be successful, there is a need for optimized antisense oligonucleotides that most efficiently achieve this result. Such optimized antisense oligonucleotides could be used alone or in conjunction with other prophylactic and/or therapeutic compositions.
Since Zamecnik & Stephenson published the first demonstration of using antisense oligonucleotides as a means to inhibit translation of viral proteins (Zemecnik and Stephenson (1978) Proc. Natl. Acad. Sci. 75: 285-288), there has been great interest in utilizing oligonucleotide-based compounds to inhibit expression of genes. These initial efforts utilized single-stranded, unmodified oligodeoxyribonucleotides or oligoribonucleotides (Agrawal et al. (1992) Ann. NY Acad. Sci. 660:2-10), which are inherently unstable in vivo, to bind to mRNA in vivo and create a double-stranded RNA template for enzymatic, or RNAse, degradation. Subsequent efforts were made to determine the utility of oligodeoxyribonucleotides that incorporated nuclease-resistant phosphorothioate and/or methylphosphonate linkages (Agrawal et. al (1988) Proc. Natl. Acad. Sci. 85:7079-7083; Metelev & Agrawal U.S. Pat. No. 5,652,355; Metelev & Agrawal U.S. Pat. No. 6,143,881; Matsukura et al. (1987) Proc. Natl. Acad. Sci. 84:7706).
Another class of RNA-based molecules that inhibit gene expression are referred to as ribozymes. Ribozymes form stem loop structures and bind to an RNA target to mediate its cleavage directly (Cech, T. (1990) Ann. Rev. Biochem. 59:543). Ribozymes selectively bind to target-RNA and catalyze a transesterification or a hydrolysis reaction to cleave specific phosphodiester linkages in single-stranded RNA. If introduced into cells, ribozymes have the potential to bind to target-mRNA and inhibit translation of such mRNA. As with single-stranded antisense technologies, the stability and activity of ribozymes have been improved through incorporation of certain chemical modifications into the ribozyme structure (Goodchild U.S. Pat. No. 6,204,027; Goodchild U.S. Pat. No. 6,573,072). While antisense oligonucleotide and ribozyme technologies continue to advance, discoveries with other oligonucleotide-based technologies are being made.
Based on the pioneering discoveries of Fire and Mello (Fire et al. (1998) Nature, 391:806-811), efforts have turned toward RNA-interfering (RNAi) technologies (e.g. short interfering-RNA (siRNA), microRNA (miRNA), DNA-directed-RNAi (ddRNAi), and single-stranded RNAi (ssRNAi)) in mammalian systems. RNAi refers to the process of post-transcriptional inhibition of gene expression using short oligonucleotides that are designed to hybridize to specific mRNA targets (Fire et al. (1998) Nature 391:806-811; Zamore et al. (2000) Cell, 101:25-33). In the case of siRNA, short, double-stranded RNA molecules utilize cellular enzymatic machinery to cleave homologous target RNA molecules. (Rana (2007) Nature Rev. Mol. Cell. Biol. 8:23-36). Double-stranded RNAi technologies rely upon administration or expression of double stranded RNA (dsRNA), which once inside the cell, is bound by an enzyme called dicer and cleaved into 21-23 nucleotides. The resulting dicer-dsRNA complex is delivered to and interacts with an Argonaut-containing complex of proteins referred to as an RNA-induced silencing complex (RISC). RISC is thought to be present in cells to catalytically break down specific mRNA molecules. Once bound by RISC, the dsRNA is unwound resulting in a ssRNA-RISC complex, which is able to hybridize to targeted mRNA. Once hybridized, the RISC complex breaks down the mRNA. In some cases, the dsRNA specific processes of dicer have been circumvented using single-stranded RNAi (ssRNAi) compositions that interact directly with RISC to achieve inhibition of gene expression (Holen et al. (2003) Nuc. Acids Res. 31:2401-2407). Although RNAi technologies are able to selectively bind to target mRNA, such molecules have also been recognized to induce non-specific immune stimulation through interaction with TLR3 (Kleinman et al., (2008) Nature 452:591-597; De Veer et. al. (2005) Immun. Cell Bio. 83:224-228; Kariko et al. (2004) J. Immunol. 172:6545-6549). This non-specific immune activation has raised questions as to the utility of RNAi technologies as pharmaceutical agents.
In the case of miRNA, a pri-miRNA is encoded in the genome of animals and plants. The pri-miRNA is a short, double stranded ribonucleic acid molecule that contains a hairpin loop. The pri-miRNA is transcribed in the nucleus and processed by the enzyme Drosha to create a double stranded, RNA molecule with a two-nucleotide overhang on its 3′ end. Once processed by Drosha, the pri-miRNA is exported into the cell's cytoplasm by a process involving Exportin-5. In the cytoplasm, the pri-miRNA is cleaved by the enzyme Dicer into shorter, double stranded segments of between 18 and 23 nucleotides, which retain the two-nucleotide overhang on the 3′ end. The resulting molecule is a mature miRNA that has antisense and sense sequences. Dicer unwinds the double stranded miRNA and one of the strands of the miRNA is incorporated into RISC, which facilitates the interaction of the miRNA with its target. The miRNA can function to regulate the expression of multiple genes by binding to the 3′-untranslated regions of specific mRNAs. In the case of plants, the miRNA may bind to a target site located within the 3′-untranslated region or within the coding region. Once bound to the target mRNA, the miRNA induces gene silencing by either mRNA degradation or inhibiting the mRNA from being translated. If the sequence is identical to the target, then the mRNA is degraded. If the sequence contains mismatched basepairs from the target, then the mRNA is inhibited from being translated. miRNAs are highly conserved and thought to be a vital component to regulating gene expression.
Although each of the antisense-based technologies has been used with some success, as a result of being based on oligonucleotides, each of these technologies has the inherent problem of being unstable in vivo and having the potential to produce off-target effects, for example unintended immune stimulation (Agrawal & Kandimalla (2004) Nature Biotech. 22:1533-1537). In the case of dsRNA, these oligonucleotides appear to have the additional issue of inefficient, in vivo delivery to cells (Medarova et. al (2007) Nature Med. 13:372-377). Despite many clinical trials of antisense oligonucleotide drug candidates, only one such compound has been approved as a drug by the FDA. This antisense compound was approved for treating CMV, but has never been marketed as a product. Additionally, no ribozyme or siRNA drug candidate has yet been approved by the FDA.
Approaches to optimizing each of these technologies have focused on addressing biostability, target recognition (e.g., cell permeability, thermostability), and in vivo activity. Often, these have represented competing considerations. For example, traditional antisense oligonucleotides utilized phosphate ester internucleotide linkages, which proved to be too biologically unstable to be effective. Thus, there was a focus on modifying antisense oligonucleotides to render them more biologically stable. Early approaches focused on modifying the internucleotide linkages to make them more resistant to degradation by cellular nucleases. These approaches led to the development of antisense oligonucleotides having a variety of non-naturally occurring internucleotide linkages, such as phosphorothioate, methylphosphonate (Sarin et al. (1988) Proc. Natl. Acad. Sci. 85:7448-7451), and peptide based linkages. (Matsukura et. al (1987) Proc. Natl. Acad. Sci. 84:7706; Agrawal et al. (1988) Proc. Natl. Acad. Sci. 85:7079-7083; Miller (1991) Bio-Technology 9:358). However, these modifications caused the molecules to decrease their target specificity and produced unwanted biological activities.
Later approaches to improve stability and retain specificity and biologic activity utilized mixed backbone oligonucleotides, which contain phosphodiester and phosphorothioate internucleotide linkages. This mixed backbone resulted in oligonucleotides that retained or improved their biological stability as compared to oligonucleotides with only phosphodiester linkages (Agrawal et al. (1990) Proc. Natl. Acad. Sci. 87:1401-1405; US Patent Publication No. 20010049436). Throughout oligonucleotide research, it has been recognized that these molecules are susceptible in vivo to degradation by exonucleases, with the primary degradation occurring from the 3′-end of the molecule (Shaw et. al (1991) Nucleic Acids Res. 19:747-750; Temsamani et al. (1993) Analytical Bioc. 215:54-58). As such, approaches to avoid this exonuclease activity have utilized (i) capping structures at the 5′ and/or 3′ termini (Tesamani et. al (1992) Ann. NY Acad. Sci. 660:318-320; Temsamani et al. (1993) Antisense Res. Dev. 3:277-284; Tang et al. (1993) Nucl. Acids Res. 20:2729-2735), (ii) linking two or more oligonucleotides through 5′-3′,5′-2,2′-3′,3′-2′ or 3′-3′ linkages between the molecules (Agrawal et al. U.S. Pat. No. 6,489,464), (iii) self-hybridizing oligonucleotides that fold back on themselves at the 3′-end, which creates a hair-pin and removes the point of access for 3′-exonuclease activity to begin (Tang et al. (1993) Nucl. Acids Res. 20:2729-2735), or (iv) incorporating RNA into the oligonucleotide molecule, thus creating an RNA/DNA hybrid molecule (Metelev at al. (1994) Bioorg. Med. Chem. Lett. 4:2929-2934; Metelev U.S. Pat. No. 5,652,355; Metelev & Agrawal U.S. Pat. No. 6,143,881; Metelev& Agrawal U.S. Pat. No. 6,346,614; Metelev & Agrawal U.S. Pat. No. 6,683,167, Metelev & Agrawal U.S. Pat. No. 7,045,609).
Other approaches to improve stability and retain specificity and biologic activity of antisense oligonucleotides utilized triplex forming, polypyrimidine oligonucleotides that bind to double-stranded DNA or RNA targets. Polypyrimidine oligonucleotides can bind to duplex DNA in the major groove through Hoogsteen hydrogen bonding and form triplex structures containing one polypurine and two polypyrimidine strands with T:A-T and C:G-C+ base triplets (Moser, H. E. and Dervan, P. B. (1987) Science 238, 645-650; Cooney, et al (1988) Science 241, 456-459). Intramolecular triplexes are also formed when the DNA homopurine and homopryrimidine strands melt and refold (Vasqueza, K. M. and Wilson, J. H. (1998) Trends Bioche. Sci. 23, 4-9). The presence of a third strand introduces severe restrictions in the flexibility of the DNA, changing its ability to recognize specific proteins along the major groove (Shields, G. C., et al. (1997) Am. Chem. Soc., 119, 7463-7469; Jimenez-Garcia, E., et al. (1998) J. Biol. Chem. 273, 24640-24648, resulting in an inhibitor of transcription and ultimately reduced gene expression. Oligonucleotides that can sequence-specifically bind to double-stranded DNA or RNA can act as transcriptional/translational regulators and offered a promising antigene/antisense strategy to control the regulation of gene expression (Giovannangeli, C. and Helene, C. (1997) Antisense Nucleic Acid Drug Dev., 413; Giovannangeli, C., et al. (1996) Biochemistry 35, 10539; Maher, L. J., et al. (1992) Biochemistry 31, 70). However, the conditions for forming stable triplexes are problematic because of limited base recognition and the non-physiologic acidic pH conditions required for protonation of cytosines in the triplex-forming oligonucleotides.
In an attempt to form such stable triplexes, polypyrimidine oligonucleotides with inverted polarity linked via a linker (i.e. one sequence having polarity 5′→3′ followed by another sequence with 3′→5′ polarity, or vice versa) have been described (Froehler, U.S. Pat. No. 5,399,676; Froehler U.S. Pat. No. 5,527,899; Froehler U.S. Pat. No. 5,721,218). In such inverted polarity oligonucleotides, the sequence on one side of the inversion binds to polypurine strand of a duplex according to the triple helix code and the sequence on the other side will bind to the adjacently located polypurine site in the opposite strand of the duplex (FIG. 1A). In this manner triple helix recognition can be extended by switching recognition from one strand of the duplex to the other and then back again, if desired and such target sequence stretch is available. In addition, these oligonucleotides may also form D-loops with the duplex as shown in FIG. 1B. In this situation, the region of the first polarity may form triplex, while the inverted portion displaces a section of one strand of the duplex to result in a substitute duplex in the relevant region. As the switchback oligonucleotides are capable of significant duplex binding activity, these oligonucleotides may be useful to inactivate the disease causing and undesirable DNA or RNA that are in duplex form. However, the composition of the molecules is limited to polypyrimidine sequences targeting polypurine sites of double-stranded RNA or DNA.
Alternatively, strategies have been developed to target single stranded DNA and RNA by triple helix formation. One of the approaches is to target polypyrimidine DNA or RNA single strands with foldback triplex-forming oligonucleotides with inverted polarity (Kandimalla, E. R., et al. (1995) J. Am. Chem. Soc. 117, 6416-6417; Kandimalla, E. R., and Agrawal, S. (1996) Biochemistry 35, 15332). In such foldback triplex-forming oligonucleotides, a polypyrimidine oligonucleotide and its complementary polypurine strand are attached through 3′-3′ attachment or 5′-5′-attachment. Such oligonucleotides containing complementary sequences attached through 3′-3′ or 5′-5′ linkages form parallel-stranded duplexes through Hoogsteen or reverse Hoogsteen base pairing. When a complementary polypyrimidine strand is available, they form triple helical structures (FIG. 2).
Despite considerable efforts, the efforts to improve the stability and maintain target recognition, without off-target effects has not generally produced oligonucleotides that would be perceived as having clinical utility. Thus, the existing antisense-based technologies leave open the challenge of creating compounds that are biologically stable, target specific, and efficient inhibitors of gene expression. New approaches are therefore needed.